Protein–protein interactions (PPIs) play a key role in most biological processes. Many of these interactions are necessary for cell survival. To understand the molecular mechanisms of biological processes, it is essential to study and characterize protein-protein interactions, identify interacting partners and protein interaction networks. There are a number of methods that have been developed to study protein-protein interactions in vitro and in vivo, such as yeast-2-hybrid, fluorescence resonance energy transfer, co-immunoprecipitation, etc. Split protein reassembly is an in vivo probe of protein interactions that circumvents some of the problems with yeast 2-hybrid (indirect interactions, false positives) and co-immunoprecipitation (loss of weak and transient interactions, decompartmentalization). Split GFP reassembly is especially attractive because the GFP chromophore forms spontaneously on protein folding in almost every cell type. However, existing split systems have limitations of evolving cellular fluorescence slowly (3-4 days), failure to evolve at all for some interactions, and also failure to work at a physiological temperature. Among different variants of GFP tested, we found that split folding-reporter GFP (frGFP, a hybrid of EGFP and GFPuv) evolves fluorescence much faster (24 - 30 h) with associating peptides and also evolves fluorescence for the RING domain BRCA1/BARD1 wild type pair. Thirty six known cancer-associated BRCA1 RING domain mutants were tested with split-frGFP system for their role in BRCA1/BARD1 interactions. Some of these mutations resulted in significant reduction of complex reassembly and cellular fluorescence.
Split frGFP fragments were further improved by directed evolution (error-prone PCR and DNA shuffling) to obtain fragments for fast and efficient fluorescence reassembly. The evolved fragments were able to generate fluorescence in as little as 12-16 h at 30 °C and in 10-14 h at 37 °C. This system was successfully tested for the detection of interactions of several therapeutically important protein pairs (such as Bcl-xL/Bim, Bcl-2/Bim, p53/hDM2, XIAP/Smac), which have key roles in apoptosis and cancer. Response to known inhibitors of these interactions was also tested using this system. These results suggest that the efficient split GFP (esGFP) fragments we developed will be very useful for in vivo screening of small molecule or cyclic peptide libraries to develop effective modulators of protein-protein interactions in their native cellular context from direct fluorescence reassembly.
Human paraoxonase-1 (huPON1) has been known for some time for its broad hydrolytic specificity against organophosphorus (OP) pesticides and nerve agents, such as, sarin, soman and tabun, etc. The large-scale expression of the soluble protein and the improvement of the stability and catalytic activity are the most critical challenges for huPON1 to be used as a drug for detoxification of OP pesticides and nerve agents. As a human protein, it is considered to be a potent candidate for the development of a catalytic bioscavenger for effective pre- and post-exposure treatment of OP intoxication. HuPON1 is very unstable and prone to aggregation when expressed in E. coli. PON1’s hydrophobic leader sequence, hydrophobic surfaces on the HDL binding sites and the lack of post-translational modifications in bacteria are considered to be some of the reasons for its lower stability in E. coli. We applied rational and semirational approaches to re-engineer huPON1 for higher expression and solubility in E. coli. At the same time, applying approaches of chaperone co-expression and MBP (maltose binding protein) fusion and optimizing purification conditions, we were able to express active, wild-type human PON1 and the engineered variants in large-scale with a high degree of purity and solubility.